Focal Segmental Glomerulosclerosis (FSGS) causes approximately 20% of nephrotic syndrome in older children and young adults. Over the past several years numerous advances have been made in our understanding of the glomerular filter on a molecular level with the identification of nephrin, podocin, and alpha-actinin 4 . Despite these advances, many patients with FSGS suffer significant morbidly (osteoporosis, sterility, cataracts, ESKD, hypertension, obesity etc) and often our treatments fail. As a result FSGS is the cause for 10% of the end stage kidney disease (ESKD) in the <20 age group (USRDS 1999). Because of the significant morbidly and cost of FSGS the NIH has sponsored clinical trial for the treatment of FSGS in children and young adults. Podocytes are the final component of the glomerular filter. As terminally differentiated cells, podocytes do not normally undergo replication. In glomerular disease, podocytes are frequently damaged and are lost into the urine. In addition, the loss of podocytes in the glomerulus (podocyte depletion) has been correlated with disease progression. We hypothesize that the amount and phenotype of the excreted podocytes will provide information on the severity of disease, response to treatment, and nature of the podocyte injury. The focus of this proposal is to develop and test a new non-invasive tool for the analysis of glomerular disease and its treatment. We have previously developed a urinary Nephrin mRNA fluorescence RT-PCR based assay for urinary podocytes. We now propose to use the urinary mRNA RT-PCR coupled with mass spectroscopy quantitation to provide attomolar sensitivity for muliplex probe sets. With this technology we will be able to precisely quantitate the amount of nephrin message in the urine (as a marker of podocyturia) and also simultaneously measure the amounts of message for multiple podocyte specific transcripts (up to 5 unique transcripts / reaction). Thus, we will be able to determine the number and transcriptional phenotype of the shed podocytes. We will use clinical samples (urine) from the NIH FSGS clinical trial to test and refine this technology. Once fully developed, this non-invasive tool will provide the clinician important information on the severity of disease, response to treatment, and nature of the podocyte injury.